The objective of this proposal is to study the molecular basis of monocyte recruitment in atherogenesis, focusing on the molecular and cell biology of a novel monocyte adhesion-associated endothelial molecule, 151-ELAM. Preliminary studies reveal a major role for 151-ELAM in binding of monocytes or monocytoid cell lines to LPS, IL-1 and TNF-alpha stimulated mouse endothelial cells (EC), to minimally modified low density lipoprotein stimulated rabbit aortic EC and to endothelium of intact aortic segments from cholesterol fed rabbits in en face binding assays. We shall explore the expression and regulation of 151-ELAM, define its importance in monocyte binding in atherogenesis, elucidate its structure, and ask whether it represents a target for therapeutic modulation of atherogenesis. 1) The expression and function of 151-ELAM in mouse and rabbit models of atherogenesis will be explored. Analysis of the patterns of expression and regulation using conventional and en face whole aorta immunohistologic approaches will be important in understanding its role in endothelial dysfunction and monocyte recruitment. Functional characterization of the molecule, in comparison with other known adhesion molecules, will be pursued in an ex vivo assay of monocyte binding to intact aortic segments from fat-fed rabbits. The effect of anti- 151-ELAM treatment on the development and progression of fatty streaks in fat-fed rabbit and apoE-deficient mouse models will be assessed to confirm its role in vivo and to evaluate its potential as a therapeutic target. 2) The role 151-ELAM plays in the multi-step process of monocyte-endothelial interaction will be assessed using flow based assays. The interaction of circulating leukocytes with endothelium at sites of extravasation involves several sequential steps: attachment, rolling, Galpha-i protein-linked activation and activation-dependent adhesion and arrest. The step or step(s) blocked by anti-151-ELAM MAbs will be assessed, thus providing critical insights into the molecular role of 151-ELAM in monocyte recruitment. 3) cDNAs encoding 151 -ELAM will be cloned and characterized by standard procedures. Sequence homologies may provide insights into 151-ELAM structure and function. Transfectants or recombinant protein will facilitate characterization of its adhesive properties and specificities. The proposed studies will elucidate the structure of this novel vascular receptor and its role in monocyte recruitment in atherogenesis. They may lead to new approaches to the regulation of the atherogenic process.
